Fermentation Optimization, Fungistatic Effects and Tomato Growth Promotion of Four Biocontrol Bacterial Strains
Round 1
Reviewer 1 Report
The manuscript describes the optimization of the fermentation process of four bacterial biocontrol agents and their effects on the growth of tomato. The authors did a lot of work and generated lots of useful data. However, given how the manuscript is written, it makes difficult to fully understand what was done as well as deciphering the key results. In my opinion, all the sections (Abstract, Introduction, M&M, Results and Discussion) need to be re-written for clarity. The authors should focus on key aspects of the study. Finally, the manuscript might require extra editorial services to improve the English grammar/syntax and minimize redundancies, making the manuscript easy to read.
Title:
The title needs to be re-phrased, for example, “Fermentation Optimization, fungistatic effects, and tomato growth promotion of four Biocontrol Bacterial strains”
Abstract:
The abstract is not informative and it is full of redundant statements. What are the two biocontrol bacteria WXCDD51 and WXCDD105? For example, Pseudomonas sp. as indicated in the text. What are the biocontrol bacteria Ba and Bs wy-1? What are the results of the initial screening in the previous stage? What is the best fermentation medium formula and conditions? What are the broad-spectrum properties tested? What are the results/effects of the single and compound strains? The concluding statement is vague and too general.
Introduction:
The introduction sections needs to be improved for clarity and cohesiveness.
Line 34-36: majorly can be re-phrased for clarity.
Line 41: the word “obvious” can be deleted.
Line 46-47: “… excessive planting density …” could be “… high planting density …”. What does continuous rain and rain …” mean? These plants are in the greenhouse and have continuous rain?
Line 50-51: Please, provide reference to support the statement that Fluvia fulva can be transmitted by seed.
Paragraphs of lines 39-47 can be merged with paragraph of lines 48-55 and re-written to minimize repetition and allow for a more coherent flow of the manuscript.
Paragraph of lines 56-62 need to be re-phrased for clarity.
Lines 63-91: This paragraph is unclear to me and needs to be re-written. Several redundant phrases make it difficult to read. For example, what is “the control effect is 27.92%”? Also, cite Ma et al. correctly, either with a number or the year.
Line 92: Again, what are the biocontrol bacteria WXCDD51, WXCDD105, Ba and Bs wy-1?
Materials and Methods:
Line 99: could be “Bacterial strains and plant material” instead of “Materials”. Lines 100-102: Please indicate a few lines to indicate previous biological control results of the strains. Line 106: please, write “LB” and “PDA” in full.
Line 102: could be “2.2. Fermentation of the four bacteria”.
Lines 111-122: These lines need to be re-phrased for good English syntax/punctuation for clarity.
Lines 126-130: How was the antibacterial activity of the sterile fermentation filtrate measured? “ … and the spore formation was observed.” seems to be redundant. What is “Take sterile water ….”? Provide a reference for the “… Oxford cup method …”
Lines 134-138: Sentences need to be re-phrased for clarity. What is the best content of each factor?
Lines 141-153: In my opinion, the authors do not have to divide section 2.2.4 into A. B. C. or D. parts. The authors could write the effects of temperature, pH, inoculum volume, or aeration as a continuous paragraph. For example, “The effect of different temperatures (20 C, 25 C, 30 C and 37 C) on the growth of the 4 bacterial strains was investigated by seeding 1 ml (2%) of …………………. into 50 ml of culture medium, in triplicates, incubated for 48h and the absorbance recorded at OD600. Also, eleven pH levels (3, 4, 5, 6, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, and 10.0) were evaluated. ………”. What is the effect of ventilation? Do you mean the effect of aeration?
Lines 154-188: This section is incoherent and difficult to read and understand what was done. For example, line 155 “Divide two of the four biocontrol bacteria into one group for a total of 6 groups.” What does mean? The whole paragraph is like this.
Lines 189-220: Please, re-write this section.
Lines 223-229: Details of the different media used to assay each of this is required here. Re-write for correct English grammar/syntax.
Lines 230-235: This section needs to be re-written for clarity.
Lines 236-239: It is unclear what the authors did here. Please, re-write so that other researchers can use your methodology to repeat the study.
Results:
-Lines 240-293: Has twelve figures as supplementary Figures. This suggests to me that these are preliminary work that should have been done before the key study of optimizing the fermentation and the effects of the biocontrol strains on growth of tomato and Botrytis and other fungi. In my opinion, it is too much for this manuscript, especially since the write-up is not well done.
-Table 1: The caption (Antimicrobial spectrum ….) of the table does not seem to describe the results presented. Also, bacteriostatic generally refers to bacteria. Could it be instead “fungistatic”? The presentation makes it confusing especially for the strain column. Please provide the strain numbers of all the fungal pathogens tested. The authors should consider having a separate column for “Host” since strawberry or rice are not strains. Please, re-write lines 314-316 to for example, “Values followed by the same letters are not significantly different at p=0.05. … ”.
Figure 1: In my opinion, this cannot be a line graph as presented because the data points are not continuous for a given treatment. If the authors prefer a line graph then the x-axis should be days after sowing (for example) and this will generate 16 line graphs representing percent seed germination for each treatment over time. Or, the current graph or data can be shown as a histogram.
-Figure 2: the same issues as Figure 1.
-Figure 3: the caption needs to be re-written.
-Lines 378-387: The authors keep indicating antibacterial rate and do not believe this is the correct terminology. It could “ inhibitory rate”. Please, write this section.
-Lines 388-400: It is not clear how the effects were quantified.
-Lines 403-411 and Figure 6 and Figure 7: The chemical pesticides pyrimetanil or polyoxin were not mentioned in M&M as treatments that it was used in the study; and suddenly they are in Figure 6 and 7.
Discussion:
The authors should consider re-writing this section to clearly discuss their results and link it previously published studies. The authors barely (Zhang et al. , line 474; Hu et al.,) cited any previous literature but seemed to reference other strains, e.g. Bacillus subtilis STO-12 but gave no citation.
Author Response
Title:
1.The title needs to be re-phrased, for example, “Fermentation Optimization, fungistatic effects, and tomato growth promotion of four Biocontrol Bacterial strains”
Thank you very much for your advice, we have made modifications to the Title.
Abstract:
2.The abstract is not informative and it is full of redundant statements. What are the two biocontrol bacteria WXCDD51 and WXCDD105? For example, Pseudomonas sp. as indicated in the text. What are the biocontrol bacteria Ba and Bs wy-1? What are the results of the initial screening in the previous stage? What is the best fermentation medium formula and conditions? What are the broad-spectrum properties tested? What are the results/effects of the single and compound strains? The concluding statement is vague and too general.
Thank you for your suggestion. There are some changes in lines 17-21. In addition, due to the excessive content of the concluding problems mentioned by the reviewer, we suggest that they be put in the conclusion at the end of the article, rather than in the abstract.
Conclusion:
- Fermentation medium formulation and fermentation conditions are as follows:
（1）WXCDD51 strain: potato starch 4.97%, yeast extract 1.48%, potassium dihydrogen phosphate 0.06%, disodium hydrogen phosphate 0.15%, sodium chloride 0.3%, magnesium sulfate 0.03%. The initial pH was 7.0, the inoculum volume was 2%, the culture temperature was 30 ºC and the liquid volume was 50 mL/250 mL.
（2）WXCDD105 strain: sucrose 4.4%, yeast extract 1.32%, 0.08% calcium chloride, 0.1%
potassium dihydrogen phosphate, 0.02% manganese sulfate. The initial pH was 6.5%, the inoculum volume was 2%, the culture temperature was 37 ºC and the liquid volume was 70 mL/250 mL.
（3）Ba strain: soluble starch 4.4%, peanut cake powder 1.31%, disodium hydrogen phosphate 0.04%, calcium chloride 0.3%, and potassium dihydrogen phosphate 0.15%. The initial pH was 6.5%, the inoculum volume was 3%, the culture temperature was 37 ºC, and the liquid volume was 30 mL/250 mL.
（4）Bs wy-1 strain: glycerin 2.2%, peanut cake powder 1.24%, calcium chloride 0.09%,
potassium dihydrogen phosphate 0.1%, disodium hydrogen phosphate 0.1%. The initial pH was 7.0,
the inoculation volume was 2%, the culture temperature was 37ºC, and the liquid volume was 70 mL/250 mL.
- Antibacterial spectrum tests showed that four biocontrol bacteria had a broad spectrum of The inhibitory rate of strain WXCDD51 against 16 diseases ranged from 33.14% to 93.74%. The strain WXCDD105 were ranged between 71.73% and 98.61%. The strain Ba was between 72.28% and 96.79%. The strain Bs wy-1 was ranged between 64.57% and 98.02%.
- Effects of single and compound strains on tomato seeds and seedlings growth. Screening out the best mixed combination S-1 (Ba+WXCDD51+WXCDD105+Bs wy-1) and S-2(Ba+WXCDD51+WXCDD105+Bs wy-1) was done. The two combinations had the best promoting
effects on promoting tomato seeds germination and tomato seeds radicle growth, respectively. The
seedlings stage tests showed that single and mixed combinations of S-1 and S-2 could significantly
promote the growth of tomato seedlings. The optimal dilution of single strains WXCDD51, WXCDD105 and the combination of S-1 and S-2 were 100 times. The optimal dilution of Ba and
Bs wy-1 were 10 times (the concentration of the original broth was 108 cfu/mL). The single-mixed
soil treatment method had the best growth-promoting effects. The combination of S-1 and S-2 using root irrigation and soaking seed treatment had the best effects, respectively. Among the single and mixed strains, the combination of S-2 had the best effect on the growth of tomato seedlings and the single strain Bs wy-1 had the worst effect. but there was non-significant difference in other treatments.
- Biocontrol effects of single and combined strains on tomato gray mold and leaf mold. The antagonistic effects of Ba+Bs wy-1 and Ba+WXCDD105 on tomato gray mold and leaf mold were
screened. The best compounding ratios for the control of gray mold combinations were Ba+WXCDD105 (2:1) and Ba+Bs wy-1 (1:3), respectively. They are named as B-1, B-2, respectively. The best compounding ratio for controlling leaf mold were Ba+Bs wy-1 (3:1) and Ba+WXCDD105 (1:4). They are named as F-1 and F-2, respectively. In vitro test, the control effects of the combination B-1 and B-2 on the gray mold and the combination of F-1 and F-2 on the leaf mold were significantly higher than those of the single strains. In the greenhouse pot experiment, the control effects of each compounding combination on gray mold and leaf mold were also significantly higher than the single strains. The preventive effect of each treatment was better than the treatment effects.
Introduction:
- The introduction sections needs to be improved for clarity and cohesiveness.
Thank you very much for your advice. We have revised this problem
4.Line 34-36: majorly can be re-phrased for clarity.
Thank you very much for your correction. Line 33 has changed the major.
5.Line 41: the word “obvious” can be deleted.
Thank you very much for your advice. We have revised this problem
6.Line 46-47: “… excessive planting density …” could be “… high planting density …”. What does continuous rain and rain …” mean? These plants are in the greenhouse and have continuous rain?
Thank you very much for your advice. We have revised this problem in Line 48 .
- Line 50-51: Please, provide reference to support the statement that Fluvia fulva can be transmitted by seed.
Thank you very much for your advice. We have revised this problem in Line 48 .
8.Paragraphs of lines 39-47 can be merged with paragraph of lines 48-55 and re-written to minimize repetition and allow for a more coherent flow of the manuscript.
Thank you very much for your advice.Lines 38 to 49, we recombine these two paragraphs.
9.Paragraph of lines 56-62 need to be re-phrased for clarity.
Thank you very much for your advice.Lines 50-56, we rewrote the paragraph.
10.Lines 63-91: This paragraph is unclear to me and needs to be re-written. Several redundant phrases make it difficult to read. For example, what is “the control effect is 27.92%”? Also, cite Ma et al. correctly, either with a number or the year.
Thank you very much for your advice.Lines 57-79, we rewrote the passage.
11.Line 92: Again, what are the biocontrol bacteria WXCDD51, WXCDD105, Ba and Bs wy-1?
Thank you very much for your advice.The modification has been completed, lines 80-81.
Materials and Methods:
12.Line 99: could be “Bacterial strains and plant material” instead of “Materials”. Lines 100-102: Please indicate a few lines to indicate previous biological control results of the strains. Line 106: please, write “LB” and “PDA” in full.
The changes have been completed, lines 88-91, 96,97.
13.Line 102: could be “2.2. Fermentation of the four bacteria”.
Thank you very much for your advice. We have revised this problem.
14.Lines 111-122: These lines need to be re-phrased for good English syntax/punctuation for clarity.
The modification has been completed, lines 101-111.
15.Lines 126-130: How was the antibacterial activity of the sterile fermentation filtrate measured? “ … and the spore formation was observed.” seems to be redundant. What is “Take sterile water ….”? Provide a reference for the “… Oxford cup method …”
The modification has been completed, lines 117-124.
16.Lines 134-138: Sentences need to be re-phrased for clarity. What is the best content of each factor?
The modification has been completed, lines 128-131.
17.Lines 141-153: In my opinion, the authors do not have to divide section 2.2.4 into A. B. C. or D. parts. The authors could write the effects of temperature, pH, inoculum volume, or aeration as a continuous paragraph. For example, “The effect of different temperatures (20 C, 25 C, 30 C and 37 C) on the growth of the 4 bacterial strains was investigated by seeding 1 ml (2%) of …………………. into 50 ml of culture medium, in triplicates, incubated for 48h and the absorbance recorded at OD600. Also, eleven pH levels (3, 4, 5, 6, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, and 10.0) were evaluated. ………”. What is the effect of ventilation? Do you mean the effect of aeration?
Lines136-143, the change has been completed, and ventilation was changed to liquid volume.
18.Lines 154-188: This section is incoherent and difficult to read and understand what was done. For example, line 155 “Divide two of the four biocontrol bacteria into one group for a total of 6 groups.” What does mean? The whole paragraph is like this.
The change has been completed, lines 145-167.
19.Lines 189-220: Please, re-write this section.
The modification has been completed, lines 185-202.
20.Lines 223-229: Details of the different media used to assay each of this is required here. Re-write for correct English grammar/syntax.
Thank you very much for your advice. We have revised this problem.The modification has been completed, lines 213-217.
21.Lines 230-235: This section needs to be re-written for clarity.
Thank you very much for your advice. We have revised this problemThe modification has been completed, lines 219-226.
22.Lines 236-239: It is unclear what the authors did here. Please, re-write so that other researchers can use your methodology to repeat the study.
Thank you very much for your advice. We have revised this problem.The modification has been completed, lines 228-231.
Results:
23.Lines 240-293: Has twelve figures as supplementary Figures. This suggests to me that these are preliminary work that should have been done before the key study of optimizing the fermentation and the effects of the biocontrol strains on growth of tomato and Botrytis and other fungi. In my opinion, it is too much for this manuscript, especially since the write-up is not well done.
Thank you very much for your advice. We have revised this problem.The research first explored the best fermentation process of the four biocontrol strains, and the optimization of the fermentation process is also part of the work content, just as the title of the article also includes this item.
24.Table 1: The caption (Antimicrobial spectrum ….) of the table does not seem to describe the results presented. Also, bacteriostatic generally refers to bacteria. Could it be instead “fungistatic”? The presentation makes it confusing especially for the strain column. Please provide the strain numbers of all the fungal pathogens tested. The authors should consider having a separate column for “Host” since strawberry or rice are not strains. Please, re-write lines 314-316 to for example, “Values followed by the same letters are not significantly different at p=0.05. … ”.
We have modified the title, and here, biocontrol bacteria all inhibit fungi,so it cannot be instead by "fungistatic". In addition, Table1 has also been modified.
- Figure 1: In my opinion, this cannot be a line graph as presented because the data points are not continuous for a given treatment. If the authors prefer a line graph then the x-axis should be days after sowing (for example) and this will generate 16 line graphs representing percent seed germination for each treatment over time. Or, the current graph or data can be shown as a histogram.
Thank you very much for your advice. We have revised this problem.
- Figure 2: the same issues as Figure 1.
Thank you very much for your advice. We have revised this problem.
- Figure 3: the caption needs to be re-written.
Thank you very much for your advice. We have revised this problem.We added the title in line 358.
28.Lines 378-387: The authors keep indicating antibacterial rate and do not believe this is the correct terminology. It could “ inhibitory rate”. Please, write this section.
Thank you very much for your advice. We have revised this problem..We revised this paragraph in lines 376-383.
29.Lines 388-400: It is not clear how the effects were quantified.
Thank you very much for your advice. We have revised this problem.The quantification of preventive effect is mentioned in the material and method. Prevention effect = (control group disease index-experimental group disease index)/control group disease index%). At the same time, a literature reference is introduced, which can be found in line 214 for details.
30.Lines 403-411 and Figure 6 and Figure 7: The chemical pesticides pyrimetanil or polyoxin were not mentioned in M&M as treatments that it was used in the study; and suddenly they are in Figure 6 and 7.
Thank you very much for your advice. We have revised this problem.We have revised it and added a few sentences, on lines 401-402,413
Discussion:
31.The authors should consider re-writing this section to clearly discuss their results and link it previously published studies. The authors barely (Zhang et al. , line 474; Hu et al.,) cited any previous literature but seemed to reference other strains, e.g. Bacillus subtilis STO-12 but gave no citation.
Thank you very much for your advice. We have revised this problem.Rewrote this paragraph, on lines 447,448,473-486.
Comments and Suggestions for Authors
Reviewer 2 Report
Line 36: The authors must consider the development of resistance by the pathogens
Line 81:The reference [24] seams to be wrong
In the following lines: 85-471-474-517 should be inserted the year and the corresponding reference
Line 124: is better to explicitate all the acronyms
Line 503: this phrase should be supported by a reference
Author Response
Line 36: The authors must consider the development of resistance by the pathogens
Thank you very much for your advice. We have revised this problem
Line 81:The reference [24] seams to be wrong
Thank you very much for your advice. We have revised this problem
In the following lines: 85-471-474-517 should be inserted the year and the corresponding reference
Thank you very much for your advice. We have revised this problem
Line 124: is better to explicitate all the acronyms
Thank you very much for your advice. We have revised this problem
Line 503: this phrase should be supported by a reference
Thank you very much for your advice. We have revised this problem
Author Response File: 
Author Response.docx
Reviewer 3 Report
In the manuscript, No. agriculture-1243483 entitled "Fermentation Optimization of Four Strains of Biocontrol Bacteria and Study on the Effect of Biocontrol on Tomato Growth" authors have optimized a liquid fermentation for four bacterial biocontrol agents and analyze their morphological, physiological, and biochemical characteristics. In the same, they have tested the plant growth-promoting activities in vitro and tested strains were studied for their impact on the growth of tomato seeds and seedlings alone and in combination. Authors have also studied disease prevention with single and combine strains. Although the study addressed an important aspect of improving plant growth and disease control in tomatoes, however, it lacks consistency and needs a better representation of results along with some new data and precise conclusions.
My specific comments are as under
Kindly provide key results in the abstract with some numerals and percentages as the present one is more about the methods only.
Kindly provide a major table for biochemical properties of four tested biocontrol agents for protease, cellulose, chitinase, glucanase, and ferritin enzymes including 16s rDNA sequence analysis results including disease prevention, and plant growth promotion results.
To improve the novelty of the research, apart from the figures, some quantitative data is needed for the preventions of tomato gray mold and leaf mold.
Kindly add the treatment details in the caption of figure numbers 1, 2, 5, 6, and 7 as it's difficult to interpret for the readers.
In figure 8, kindly explain what particular secretory substances in strain WXCDD51 and WXCDD105 under figure 8a and 8b. It’s really, confusing in the present form as in the text figure 8a and 8b is for HCN production whereas the figure it is showing the protease result.
In such type of study, it is must to highlight the key findings under the separate conclusion section with to the point’s recommendations from the studies strains as single and combine effects have been studied. It’s lacking in the present MS.
Author Response
Thank you very much for your suggestions. We spent some time to revise most of this article, please have a look. Your suggestions are very important to me, and I hope you can correct my criticism.We have revised the references, the pictures, the details, and the whole article
Round 2
Reviewer 1 Report
The authors have added more information and done some revisions but the manuscript is still difficult to read and scientifically incoherent due to several redundant statements. English grammar/syntax needs significant improvement. All the sections need to be thoroughly revised for clarity. The authors should consider using an English editing service. Also, for clarity, any product that inhibits the growth of bacteria could be referred to as having a bacteriostatic effect while fungi-inhibiting products are fungistatic. It seems the authors misuse this terms in the manuscripts. Also, is pyrimethanil a bactericide or a fungicide.
Author Response
Thank you very much for your advice. We have corrected the grammar of the article and polished it.Pyrimethanil is a fungicide.
Author Response File: Author Response.docx
Reviewer 3 Report
The authors have revised the manuscript "agriculture-1243483". The MS can be improved further by separating the conclusion section and pinpointing the take-home findings.
Author Response
Thank you for your suggestions.We have revised this issue and added a conclusion section.
